1. Field of the Invention
The present invention relates to a process for creating transgenic Eucalyptus plants comprising infecting adventitious shoots derived from mature trees of Eucalyptus plants with Agrobacterium tumefaciens.
2. Related Art
Eucalyptus plants are polygenus plants comprising more than 500 species that are native to the Oceania region and predominantly Australia. Many of these Eucalyptus plants have excellent growth properties, the ability to adapt to various environments, and a low level of serious insect damage. Since they are also suited industrially to the production of lumber, pulp and firewood, afforestation of Eucalyptus species plants is conducted in various regions around the world. According to a survey conducted by The United Nations Food and Agriculture Organization (FAO) in 1990, Eucalyptus plants are planted over an estimated area of 10 million hectares throughout the world, accounting for approximately one-fourth of the artificial forest area in tropical regions. In order to further enhance the productivity of these Eucalyptus plants, breeding is conducted by selection and hybridization. In Brazil, for example, breeding has been performed by selection and hybridization targeting to enhancement of basic density and pulp yield, which are two important factors in the paper pulp industry, in addition to growth properties, and yield has been reported to be increased by 2.3 times in terms of the basic density and by 2.4 times in terms of the pulp yield.
Moreover, in recent years, specific gene isolation, modification and reinsertion of recombinant genes into plants has become possible in numerous plant species due to the progress of gene manipulation technology, and plant breeding is being aggressively conducted involving transformation of useful genes isolated from heterogeneous organisms or modified genes of genes inherently retained by the plant itself, which was not possible with conventional breeding methods such as selection and hybridization breeding. Numerous examples of such breeding are reported in the literature, including breeding of loblolly pine (Sederoff, et al., Bio/Technology 4: 647-649 (1986)), poplar (Fillatti et al., Mol. Gen. Genet. 206: 192-199 (1987)), walnut (McGranahan, et al., Bio/Technology 6: 800-804 (1988)), apple (James, et al., Plant Cell Rep. 7; 658-661 ) (1989)) and plum (Mante, et al., Bio/Technology 9: 853-857 (1991)).
However, not all woody plants are able to be transformed with stability at the present time. In order to establish transformation technology using Agrobacterium tumefaciens in the target woody plant, it is necessary to develop (1) a method for regenerating the plants from a transformed cell, and (2) a method for infecting Agrobacterium tumefaciens into the plant tissue. In order to accomplish this, the inventors of the present invention have developed methods of regenerating plants from tissue or isolated cells of Eucalyptus plants by the utilization of shoot primordia (Japanese Unexamined Patent Publications Nos. 62-55020, 63-7720, 64-47318, 2-265419, 4-4828, 5-236832, 9-98684, UK Patent No. GB2195656B; U.S. Pat. No. 5,310,673), the utilization of protoplasts (Japanese Unexamined Patent Publications Nos. 2-128631, 64-43138, 2-128631, UK Patent No. GB2231585B; U.S. Pat. No. 5,310,673), and the utilization of precocious branches (Japanese Unexamined Patent Publication No. 10-304785). Moreover, the inventors of the present invention have proposed a method of producing transgenic plants comprising introducing a foreign gene by electroporation into the protoplast of a Eucalyptus plant aiming the transformation using these regeneration methods (Japanese Unexamined Patent Publication No. 4-53429).
However, in the case of producing transformants by electroporation, not only is considerably time required until a transformant is obtained, but there is also the problem of transformants being obtained at a low frequency. Therefore, a method was proposed for efficiently obtaining transgenic plants by infecting the cotyledon or hypocotyl derived from seedlings of Eucalyptus plants with Agrobacterium tumefaciens (Japanese Unexamined Patent Publications Nos. 7-203790 and 8-89113).
These methods made it possible to transform Eucalyptus plants for which transformation was previously difficult. However, in the case of infecting segments of the explant from mature trees growing outdoors with Agrobacterium tumefaciens using the same methods, problems occurred that included blackening of segments of the explant by polyphenol induced as a result of wounding or co-culturing with Agrobacterium tumefaciens, thereby preventing the production of transgenic plants.
On the other hand, with respect to transformation of Eucalyptus plants with Agrobacterium bacteria by persons other than the inventors of the present invention, although the successful gene insertion has been previously reported in Eucalyptus globulus, regeneration of the transgenic plant was not performed (Landre, et al., Plant Sci. 127: 81-91 (1997); Serrano, et al., J. Exp. Botany 47: 285-290 (1996)). In addition, although successful regeneration of the transgenic plant has been reported in Eucalyptus camaldulensis, due to the low transformation efficiency, this did not lead to a stable transformation system, (Mullins, et al., Plant Cell Rep. 16: 787-791 (1997)). Moreover, although it has also been reported again in Eucalyptus camaldulensis that a transgenic plant was successfully regenerated using the hypocotyl for the transgenic material, since the hypocotyl is used for the transformation material, transformation is not possible for a specific mature tree (Ho, et al., Plant Cell Rep. 17: 675-680 (1998)). Moreover, a method is proposed in International Application WO96/25504 for the purpose of transforming mature trees in which segments of the explant are removed from a mature tree of a Eucalyptus plant followed by tissue culturing and transformation to the resulting plant.
Although the prior experiments enabled the transformation of a mature tree of a eucalyptus plant, the formation rate of transgenic calli and the regeneration rate of transgenic plants were inadequate.
As a result of various studies to solve the above problems, the inventors of the present invention found that mature tree of Eucalyptus plants can be efficiently transformed by inducing tissue cultures having high transformation ability from mature trees of Eucalyptus plants.
Accordingly, the present invention provides a process for producing transgenic plants by inducing adventitious shoots that can be efficiently transformed from a mature tree of Eucalyptus plant, infecting the adventitious shoots with Agrobacterium tumefaciens, forming transgenic calli from the infected adventitious shoot, forming shoot primordia from the transgenic calli and regenerating transgenic plants from the shoot primordia.
More specifically the present invention provides a process for production of transgenic Eucalyptus plants from a mature tree of Eucalyptus plant, comprising the steps of:
(1) preparing a shoot tip explant from a mature tree of Eucalyptus plant;
(2) inducing the shoot tip explant to form adventitious shoots;
(3) pre-culturing the adventitious shoots to prepare infection in an infection induction medium;
(4) infecting segments of explant cut out from the pre-cultured adventitious shoots with Agrobacterium tumefaciens in an infection medium;
(5) rotary-culturing the segments of the explant from the step (4) in a medium for sterilization containing antibiotics so as to sterilize the Agrobacterium tumefaciens attached to the segments of the explant, to form calli and to select transformed calli;
(6) forming shoot primordia from the transformed calli; and
(7) regenerating transgenic plants from the transformed calli.
In preferred embodiments, the adventitious shoots infected by Agrobacterium tumefaciens are precocious branches or axillary shoots, and preferably precocious branches.
In a preferred embodiment, the infection in the step (4) is carried out by infecting the adventitious shoots with Agrobacterium tumefaciens after pre-culturing in the step (3) in an infection induction medium containing 0.01 to 2 mg/1 of naphthaleneacetic acid (NAA) for 7 to 21 days under dark conditions.
According to a preferred embodiment of the present invention, the infection medium used in the step (4) contains 0.2 to 5.0 mg/l of naphthaleneacetic acid (NAA) and 0.02 to 1.0 mg/l of 1-(2-chloro-4-pyridyl)-3-phenylurea or N-(2-chloro-4-pyridyl)-Nxe2x80x2-phenylurea (4-PU or 4CPUU).
According to a preferred embodiment of the present invention, the eucalyptus plant is Eucalyptus camaldulensis, Eucalyptus globulus, Eucalyptus grandis, Eucalyptus urophylla, or a hybrid derived from one of them as maternal or paternal tree.